The DNA sequencing methods of choice for nearly all scientific and commercial applications are based on the dideoxy chain termination approach pioneered by Sanger, e.g. Sanger et al, Proc. Natl. Acad. Sci., 74: 5463-5467 (1977). The method has been improved in several ways and, in a variety of forms, is used in all commercial DNA sequencing instruments, e.g. Hunkapiller et al, Science, 254: 59-67 (1991).
The chain termination method requires the generation of one or more sets of labeled DNA fragments, each having a common origin and each terminating with a known base. The set or sets of fragments must then be separated by size to obtain sequence information. The size separation is usually accomplished by high resolution gel electrophoresis, which must have the capacity of distinguishing very large fragments differing in size by no more than a single nucleotide. Despite many significant improvements, such as separations with capillary arrays and the use of non-gel electrophoretic separation mediums, the technique does not readily lend itself to miniaturization or to massively parallel implementation.
As an alternative to the Sanger-based approaches to DNA sequencing, several so-called "base-by-base" or "single base" sequencing approaches have been explored, e.g. Cheeseman, U.S. Pat. No. 5,302,509; Tsien et al, International application WO 91/06678; Rosenthal et al, International application WO 93/21340; Canard et al, Gene, 148: 1-6(1994); and Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994). These approaches are characterized by the determination of a single nucleotide per cycle of chemical or biochemical operations and no requirement of a separation step. Thus, if they could be implemented as conceived, "base-by-base" approaches promise the possibility of carrying out many thousands of sequencing reactions in parallel, for example, on target polynucleotides attached to microparticles or on solid phase arrays, e.g. International patent application PCT/US95/12678.
Unfortunately, "base-by-base" sequencing schemes have not had widespread application because of numerous problems, such as inefficient chemistries which prevent determination of any more than a few nucleotides in a complete sequencing operation. Moreover, in base-by-base approaches that require enzymatic manipulations, further problems arise with instrumentation used for automated processing. When a series of enzymatic steps are carried out in reaction chambers having high surface-to-volume ratios and narrow channel dimensions, enzymes may stick to surface components making washes and successive processing steps very difficult. The accumulation of protein also affects molecular reporter systems, particularly those employing fluorescent labels, and renders the interpretation of measurements based on such systems difficult and inconvenient. These and similar difficulties have significantly slowed the application of "base-by-base" sequencing schemes to parallel sequencing efforts.
An important advance in base-by-base sequencing technology could be made, especially in automated systems, if an alternative approach was available for determining the terminal nucleotides of polynucleotides that minimized or eliminated repetitive processing cycles employing multiple enzymes.